Assessment of Antibody and T-Cell Responses to the SARS-CoV-2 Virus and Omicron Variant in Unvaccinated Individuals Recovered From COVID-19 Infection in Wuhan, China

This cohort study examines immune system responses to the Omicron strain of the SARS-CoV-2 virus among unvaccinated individuals in Wuhan, China, who recovered from infection with the initial strain of the virus.

study. 1 The study included two groups. In group one, 113 plasma samples were taken from 113 recovered patients to perform ELISA assay, in which a total of 40 plasmas were further used for pseudovirus neutralization assays. In group two, PBMCs were collected from 41 individuals and analyzed by ELISpot to detect the SARS-CoV-2-specific memory T cell responses.

Inclusion and exclusion criteria Inclusion criteria:
All patients with laboratory confirmed COVID-19 who were discharged from Wuhan Research Center for Communicable Disease Diagnosis and Treatment, Chinese Academy of Medical Sciences (Wuhan, China) between Jan 7 and May 29, 2020.

Exclusion criteria
(1) those who died before the follow-up visit, (2) those for whom follow-up would be difficult owing to psychotic disorder, dementia, or readmission to hospital attributed to underlying diseases, (3) those who were unable to move freely due to concomitant osteoarthropathy or immobile before or after discharge due to diseases such as stroke or pulmonary embolism, (4) those who declined to participate, (5) those unable to be contacted, and (6) those living outside of Wuhan or in nursing or welfare homes.

Plasma and PMBC isolation
Venous blood was collected from participants and processed within 12 h to isolate plasma and © 2022 Guo L et al. JAMA Network Open.
peripheral blood mononuclear cell (PBMCs). Plasma was separated by centrifugation at 300 x g for 10 minutes and stored at -80°C until testing. PBMCs were isolated from blood using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL) according to the manufacturer's instructions.

Peptide synthesis
Peptides covering the mutated regions of S, N, M, and E protein in Omicron and the corresponding Wuhan strain control pool containing homologous peptides were synthesized (purity >90%; Sangon Biotech, Shanghai, China) (eTable 1, eTable 2).

Enzyme-linked immunosorbent assay (ELISA)
IgG antibody titres against the spike protein (S) and receptor binding domain (RBD) of SARS-CoV-2 Wuhan strain and Omicron strain were evaluated using the enzyme-linked spot of the negative control and ≥10 s.f.u./10 6 PBMCs. If negative control wells had >100 s.f.u. /10 6 PBMC or positive control wells had <1000 s.f.u/10 6 PBMC, the results were excluded from further analysis.

Outcomes
The primary outcomes were NAb titers and T cell responses. The titers of SARS-CoV-2specific NAbs were expressed as a 50% half-maximal inhibitory does (ID50). T cell responses were expressed as magnitude of IFN-γ produced by SARS-CoV-2 specific-T cells. The secondary outcomes included S-, RBD-IgG, which were expressed as optical density at 450 nm (OD 405); demographic features of recovered patients, including age, sex, and days after infection.

Ethics approval
The study was approved by the Institutional Review Boards of Wuhan Research Center for Communicable Disease Diagnosis and Treatment, Chinese Academy of Medical Sciences (KY-2020-80.01). Written informed consent was obtained from each COVID-19 patient.

Statistical analysis
The comparison of IgG seropositivity was done with χ² test. Multiple comparisons of neutralising antibody titers among Wuhan strain and Beta, Delta, and Omicron variants were performed using the Kruskal-Wallis test followed by a post hoc Dunn's correction. Paired plasma antibody titers and T cell responses were compared using a two-tailed Wilcoxon matched-pairs signedrank test. A two-sided P < .05 was considered to be statistically significant. All statistical analysis was conducted using GraphPad Prism 9.3.0 (GraphPad Software, San Diego, CA).